plants Brief Report Loquat (Eriobotrya japonica) Is a New Natural Host of Apple Stem Pitting Virus Félix Morán , Celia Canales, Antonio Olmos and Ana Belén Ruiz-García * Centro de Protección Vegetal y Biotecnología, Instituto Valenciano de Investigaciones Agrarias (IVIA), Ctra. Moncada-Náquera km 4.5, Moncada, 46113 Valencia, Spain; [email protected] (F.M.); [email protected] (C.C.); [email protected] (A.O.) * Correspondence: [email protected] Received: 29 September 2020; Accepted: 11 November 2020; Published: 13 November 2020 Abstract: Loquat (Eriobotrya japonica) is a minor but important woody crop cultivated in Asia and Europe. High-throughput sequencing (HTS) analysis of an asymptomatic loquat plant using RNAseq Illumina technology has allowed the detection for the first time of apple stem pitting virus (ASPV), the type species of the genus Foveavirus in the family Betaflexiviridae, infecting this crop. A nearly complete genome of 9303 nts (ASPV-SL61) reconstructed bioinformatically shows the typical genomic structure of this viral species and a highest nucleotide identity (85.9%) with the Chinese ASPV isolate YLX from pear. A close phylogenetic relationship between ASPV-SL61 and ASPV-YLX has been confirmed by the sequence analysis of full-length ASPV genomic sequences available in the databases. In fact, a phylogenetic study based on a partial CP N-terminal sequence previously proposed to be involved in host adaptation has shown that ASPV-SL61 loquat isolate is more closely related to ASPV pear isolates. The presence of ASPV in loquat has been further confirmed by RT-PCR and Sanger sequencing and DAS-ELISA. An incidence of 15% was determined in one of the loquat Spanish growing areas. The sequence analysis of the partial CP sequences amplified by RT-PCR has shown a high level of variability between loquat isolates. To our knowledge, this is the first record of loquat as a natural host of ASPV. Keywords: ASPV; HTS; loquat 1. Introduction Apple stem pitting virus (ASPV) is the type species of the genus Foveavirus, family Betaflexiviridae, subfamily Quinvirinae [1]. ASPV has been reported to be widely distributed and to infect several hosts including apple, pear, hawthorn, quince, cherry, and nanking cherry [2–5]. Although ASPV has been related to a broad range of symptoms such as green crinkle, star crack, epinasty, decline and stem pitting in apple, vein yellowing, leaf red mottling and necrotic spotting in pear, and fruit deformations in quince, its infection has frequently been reported to remain asymptomatic [6–8]. Several studies have shown a high degree of intraspecies diversity for ASPV [6,8,9]. This genetic diversity has been proposed to be host-related and to play a role in new host adaptation [8–10]. Loquat (Eriobotrya japonica), which belongs to the family Rosaceae, is a tree native to China cultivated in Asia and Europe and appreciated for its early sweet fruit and medicinal properties [11]. Spain is the main loquat exporter with an annual production of 40,000 tones representing a valuable local economic income in some regions of the country [12]. To date, only three viral pathogens have been reported to infect loquat, apple chlorotic leafspot virus (ACLSV), apple stem grooving virus (ASGV), and loquat virus A (LoVA) [11,13]. Although none of these viruses have been clearly associated with any particular disease or reduced fruit production, emergence of new viral pathogens and/or synergism between different viral species might negatively affect this crop. Plants 2020, 9, 1560; doi:10.3390/plants9111560 www.mdpi.com/journal/plants Plants 2020, 9, x FOR PEER REVIEW 2 of 9 Plantsassociated2020, 9 ,with 1560 any particular disease or reduced fruit production, emergence of new viral pathogens2 of 9 and/or synergism between different viral species might negatively affect this crop. With the aim of preventing the eventual emergence of viral diseases in loquat, several trees from a loquat growing area in the eastern region of Spain were analyzedanalyzed by high-throughput sequencing (HTS), RT-PCR, andand DAS-ELISA.DAS-ELISA. ThisThis studystudy hashas allowed the identificationidentification for the firstfirst time of apple stem pitting virus (ASPV) infecting loquat, providing new insights into the host range of this viral pathogen and contributing to a betterbetter understandingunderstanding ofof thethe sanitarysanitary statusstatus ofof thisthis crop.crop. 2. Results HTS analysis on total RNA extracted from an asymptomatic loquat plant (SL61, cv. Algerie) from a privateprivate gardengarden in in Segorbe, Segorbe, Spain, Spain, yielded yielded 33,340,022 33,340,022 reads reads (144.2 (144.2 nts nts average average size) size) after after trimming trimming and qualityand quality control. control. A host A genomehost genome subtraction subtraction step wasstep performed,was performed, and the and 3,251,340 the 3,251,340 remaining remaining reads subjectedreads subjected to de novo to de assembly. novo assembly. BLASTN BLASTN/X/X analysis an ofalysis the 18,908 of the contigs 18,908 obtainedcontigs obtained revealed revealed the presence the ofpresence 1 long of contig 1 long related contig to related ASPV. to The ASPV. contig The of cont 9303ig nt of in 9303 length nt in was length covered was bycovered 6622 readsby 6622 with reads an averagewith an coverageaverage coverage of 106.5x of and 106.5x comprised and comprised the full-length the full-length coding sequence coding assequence well as 45as ntwell 5’UTR as 45 and nt 1235’UTR nt 3and0UTR 123 regions nt 3′UTR (ASPV-SL61, regions (ASPV-SL61, accession numberaccession MW045213). number MW045213). The isolate The ASPV-SL61 isolate ASPV-SL61 showed 85.9%showed nucleotide 85.9% nucleotide identity identity with the with Chinese the Chinese isolate YLX isolate from YLX pear from (KY798310) pear (KY798310) and shared and theshared typical the genometypical genome structure structure of other of foveaviruses other foveaviruses (Figure (Figure1) encoding 1) encoding five ORFs, five theORFs, RNA the dependent RNA dependent RNA polymerase,RNA polymerase, RdRp RdRp (2183 aa),(2183 the aa), triple the genetriple blockgene proteins,block proteins, TGB1 TGB1 (224 aa), (224 TGB2 aa), TGB2 (121 aa), (121 and aa), TGB3 and (71TGB3 aa) (71 and aa) the and coat the coat protein, protein, CP (413CP (413 aa). aa). The Th aminoe amino acid acid identity identity percentage percentage observedobserved between ASPV-SL61 and ASPV-YLX at the didifferentfferent ORFsORFs werewere 92.9% (RdRp), 98.2% (TGB1), 93.3% (TGB2), 97.2% (TGB3), and 87.7% (CP). The BLASTN/X BLASTN/X analysis performed did not show the presence in SL61 of any other viral-related sequences. Figure 1. Genome structure of ASPV-SL61. Sequence length and genomic positions of the five ORFs Figure 1. Genome structure of ASPV-SL61. Sequence length and genomic positions of the five ORFs (RdRp, TGB1, TGB2, TGB3, and CP), as well as the 50 and 30 UTR are indicated. (RdRp, TGB1, TGB2, TGB3, and CP), as well as the 5′ and 3′ UTR are indicated. A phylogenetic analysis based on full-length sequences available in the databases showed that ASPV-SL61A phylogenetic isolate is analysis phylogenetically based on related full-length to pear sequences isolates ofavailable China (isolatein the databases YLX) and showed South Korea that (isolateASPV-SL61 WH, isolate LC475151) is phylogenetically with high bootstrap related to support pear isolates (Figure of2 ),China in agreement (isolate YLX) with and the South nucleotide Korea similarity(isolate WH, observed LC475151) between with SL61 high and bootstrap YLX by support BLASTN (Figure/X analysis. 2), in agreement with the nucleotide similarityA subsequent observed sequence between analysis SL61 and using YLX a 250by BLASTN/X aa CP partial analysis. sequence corresponding to the N-terminal regionA ofsubsequent the coat protein sequence from analysis 303 di ffusingerent ASPVa 250 aa isolates CP partial available sequence in the corresponding databases, most to of the them N- fromterminal apple region and pear of the (Table coat S1),protein as well from as ASPV-SL61303 different was ASPV performed isolates (Figure available2). Thisin the part databases, of the CP most has beenof them described from apple to be and the pear most (Table variable S1), region as well of as the ASPV-SL61 protein and was it isperformed supposed (Figure to be involved 2). This inpart host of adaptationthe CP has [8been,10]. described The phylogenetic to be the tree most constructed variable basedregion on of this the N-terminal protein and CP it sequence is supposed alignment to be showedinvolved that in host ASPV adaptation isolates are [8,10] predominantly. The phylogenetic grouped tree by host constructed as previously based described on this [N-terminal8,9]. ASPV CP N-terminalsequence alignment region grouped showed in that two AS clusters,PV isolates one correspondingare predominantly to the grouped majority by of host isolates as previously infecting appledescribed and [8,9]. a second ASPV one CP related N-terminal to most region of the isolatesgrouped infecting in two clusters, pear, although one corresponding some apple isolates to the clusteredmajority of with isolates pear isolatesinfecting and apple vice versa.and a Isolatesecond SL61 one fromrelated loquat to most grouped of the into isolates the main infecting pear cluster. pear, Severalalthough deletions some apple on the isolates N-terminal clustered region with of the pear CP isolates respect and to the vice isolate versa. PA66 Isolate (NC_003462) SL61 from have loquat also beengrouped involved into the in ASPVmain pear host adaptationcluster. Several [10]. deletions Three deletions on the (3,N-terminal 5, and 1 nts) region and of one the insertion CP respect (3 nts) to havethe isolate been foundPA66 (NC_003462) in this genomic have region also been of ASPV-SL61. involved in ASPV host adaptation [10]. Three deletions (3, 5, and 1 nts) and one insertion (3 nts) have been found in this genomic region of ASPV-SL61.